Dysfunction of the Na current (INa) flowing through the a subunit of the cardiac Na channel encoded by SCN5A participates in pathogenic mechanisms for arrhythmia, heart failure, and ischemia. The mechanism for the clinical phenotype of a particular genotype comes from the underlying molecular phenotype (e.g., INa amplitude and kinetics) and cellular phenotype (e.g., action potentials) caused by the mutation. In the previous period we focused on the molecular phenotype in heterologous expression systems associated with mutations of SCN5A. We discovered a ubiquitous human splice variant (Q1077del) that affects function of other mutations, clinically relevant functional effects of 8 common polymorphisms (e.g. H558R), and drug rescue of trafficking defective mutations. We also showed a novel dominant negative effect of one Brugada syndrome SCN5A allele on another. Two aims (1 and 3) of this proposal follow up on these pathogenic mechanisms of genetic variation in SCN5A. Two additional aims (2 and 4) take the project in a new direction. It is increasingly clear that INa is a function of the SCN5A macromolecular complex where more than one a subunit combines with [unreadable] subunits and other channel interacting proteins (ChIPs) such as caveolin and syntrophins. Pathogenic genotypes have yet to be discovered in many patients from cohorts with suspected inherited arrhythmia syndromes (LQTS, SIDS, SUDS). From patients in these cohorts we have recently discovered mutations in putative ChIPs (CAV3, SNTA1, GPD1L, SCN4B) and shown that they affect INa in a way that may be pathogenic. In the next period we will continue a recently implemented more integrative approach to the study by expressing mutations in myocardial cells. This more complete cardiac environment allows for characterization of both the cellular phenotype and the molecular phenotype under relevant conditions such as adrenergic state in the presence of the intact macromolecular complex. In Aim 1 we will discover pathogenic mechanisms involving an SCN5A variant by characterizing the molecular and cellular phenotypes. In Aim 2 we will discover mutations in novel genes encoding putative SCN5A ChIPs from patients with suspected inherited arrhythmia and no known pathogenic genotype, and define molecular and cellular phenotypes to establish an INa mediated mechanism. In Aim 3 we will investigate SCN5A a-a subunit interactions (such as dominant negative and rescue effects). In Aim 4 we will address molecular mechanisms accounting for the molecular phenotype of altered INa for two novel arrhythmia genes (SNTA1 and GPD1L). The integrated approach of this proposal takes into account the SCN5A macromolecular complex in more native systems to determine both molecular and cellular phenotypes, and molecular mechanisms underlying the molecular phenotype. In addition to better understanding of the SCN5A macromolecular complex, the expected results should yield insight into pathogenetic mechanisms of the genotype-phenotype link for inherited and acquired arrhythmia and other cardiac disease. PUBLIC HEALTH RELEVANCE: Inherited mutations in the gene that encodes a membrane ion channel protein called SCN5A cause injury and death from arrhythmia in syndromes such as Sudden Infant Death Syndrome (SIDS), Sudden Unexpected Death Syndrome (SUDS), and long QT syndrome (LQTS). This project will discover mutations in novel genes (not SCN5A but related) discovered in patients with these syndromes and how they interact with SCN5A to alter function and cause arrhythmia. The results are expected to improve diagnosis of these inherited syndromes and suggest ways to treat and prevent them.